Temperature sensitive plasmids of P. haemolytica

ABSTRACT

Mutants of  P. haemolytica  provide excellent safety and efficacy when used as vaccines in ruminants, for example cattle, sheep, and goats, subject to pneumonic pasteurellosis. They can be administered by a variety of routes. Especially preferred is the use in animal feeds. The mutants are not reverting and contain no foreign DNA and no introduced antibiotic resistance genes.

This application is a division of co-pending Ser. No. 09/160,340 filedSep. 25, 1998, now U.S. Pat. No. 6,331,303, which claims the benefit ofco-pending provisional application Serial No. 60/060,060, filed Sep. 25,1997. Both applications are incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

This invention is related to the field of bacterial genetics and moreparticularly to the field of respiratory pathogens of farm animals.

BACKGROUND OF THE INVENTION

P. haemolytica as a pathogen causes serious economic damage to theanimal farming industry. Vaccines which have been developed in an effortto control the disease have met with variable but limited success.Because the disease is caused in significant part by the animals' ownreaction to P. haemolytica infection, inappropriately designed vaccinesmay actually worsen the clinical condition of infected vaccinates. Thus,there is a continuing need in the art for safe and effective vaccineswhich can reduce the morbidity and/or mortality of ruminants due to P.haemolytica.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a P. haemolyticabacterium useful as a vaccine strain.

It is another object of the present invention to provide a method ofinducing immunity to pneumonic pasteurellosis in ruminants.

It is an object of the present invention to provide a vaccine strainagainst pneumonic pasteurellosis.

Another object of the invention is to provide a ruminant feed.

Another object of the invention is to provide a temperature sensitiveplasmid for manipulation of P. haemolytica.

These and other objects of the invention are achieved by one or more ofthe embodiments described below. One embodiment of the inventionprovides a P. haemolytica bacterium which expresses no biologicallyactive leukotoxin, expresses a form of leukotoxin molecule which inducesantibodies which specifically bind to leukotoxin, and contains noforeign DNA.

Another embodiment of the invention provides a method of inducingimmunity to pneumonic pasteurellosis in ruminants. A bacterium isadministered to a ruminant. Immunity to the bacterium is therebyinduced. The bacterium expresses no biologically active leukotoxin,expresses a form of leukotoxin molecule which induces antibodies whichspecifically bind to leukotoxin, and contains no foreign DNA.

Yet another embodiment of the invention provides a feed for ruminants.The feed comprises a bacterium which expresses no biologically activeleukotoxin, expresses a form of leukotoxin molecule which inducesantibodies which specifically bind to leukotoxin, and contains noforeign DNA.

Even another embodiment of the invention provides a vaccine for reducingmorbidity in ruminants. The vaccine comprises a P. haemolytica bacteriumwhich expresses no biologically active leukotoxin, expresses a form ofleukotoxin molecule which induces antibodies which specifically bind toleukotoxin, and contains no foreign DNA.

Still another embodiment of the invention provides a temperaturesensitive plasmid. The plasmid replicates at 30° C. but not at 40° C. inP. haemolytica. Moreover, it is of the same incompatibility group as theplasmid which has been deposited at the ATCC with Accession No. 98895.

The present invention thus provides the art with tools for geneticallymanipulating an agriculturally important pathogen. It also providesuseful mutant strains which can be used effectively to reduce morbidityamong ruminants, such as cattle, sheep, and goats, due to Pasteurellahaemolytica.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Fate of temperature-sensitive plasmid in Pasteurella hemolyticaafter passage at 30° C. and 40° C.

FIG. 2. The Pasteurella hemolytica leukotoxin operon with 3.15 kb EcoRVfragment. lktC, acylates leukotoxin structural gene to activate; LktA,leukotoxin structural gene; lktB/D, involved in leader-independentleukotoxin export.

FIG. 3. In-frame deletion of 3.15 kb EcoRV fragment of lktCA using NaeI.

FIG. 4. Integration of replacement plasmid into chromosome.

FIG. 5. Resolution of replacement plasmid from chromosome.

FIG. 6. Western blot of native leukotoxin and ΔlktA using anti Lktmonoclonal antibody.

DETAILED DESCRIPTION

It is a discovery of the present invention that a non-reverting mutantof P. haemolytica which expresses a mutant form of leukotoxin is usefulas a vaccine. Moreover, this mutant has been found to be useful whenadministered to the tonsils, via the oral route, and via the nasalroute. Thus extremely inexpensive and easy methods of vaccinatinganimals can be accomplished, simply by top dressing animal feed.

The mutant preferably is a deletion mutant. One such mutant leukotoxinprotein made is about 66 kD, although other such mutants can be used, solong as the protein is long enough to be immunogenic, preferably atleast 10, 15, or 20 amino acids long. It is believed that a longerdeleted molecule is preferred to achieve a strong immune response. It ispreferred that the mutant bacterium contains no exogenous genes, such asdrug resistance genes, which can cause environmental and health problemsif not contained. In addition, it is preferred that the mutation be anon-reverting mutation, such as a deletion mutation.

Mutant forms of leukotoxin of the present invention induce antibodieswhich specifically bind to leukotoxin. Antibodies which specificallybind to leukotoxin provide a detection signal at least 2-, 5-, 10-, or20-fold higher than a detection signal provided with proteins other thanleukotoxin when used in Western blots or other immunochemical assays.Preferably, antibodies which specifically bind to leukotoxin do notdetect other proteins in immunochemical assays and can immunoprecipitateleukotoxin from solution. More preferably, the antibodies can bedetected in an indirect hemagglutination assay and can neutralizeleukotoxin.

Although the oral route is preferred for ease of delivery, other routesfor vaccination can also be used. These include without limitation,subcutaneous, intramuscular, intravenous, intradermal, intranasal,intrabronchial, etc. The vaccine can be given alone or as a component ofa polyvalent vaccine, i.e., in combination with other vaccines.

Also provided by the present invention is a temperature sensitiveplasmid which replicates at 30° C. but not at 40° C. in P. haemolytica.Preferably the plasmid is of the same incompatibility group as pD80,i.e., it shares the same origin of replication. One such plasmid wasdeposited on Sep. 25, 1998 at the ATCC, now located at 10801 UniversityBoulevard, Manassas, Va. 20110-2209, under Accession No. 98895.

Vaccination with modified-live combination of P. haemolytica serotypes 5and 6 protects against combined homologous virulent challenge extremelywell, based on clinical signs, postmortem lesions, and results ofbacterial culture. Animals which have been so vaccinated remain active,alert, afebrile, and on-feed. The vaccine can not only prevent death dueto P. haemolytica, but can also reduce symptoms of pneumonicpasteurellosis, such as lung lesion volume, fever, decreased appetite,loss of lung ventilation capacity, fibrinous pleural effusion and/oradhesions, bacterial load, and depression.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples which are provided herein for purposes of illustrationonly, and are not intended to limit the scope of the invention.

EXAMPLE 1 Modified-live Oral and Parenteral Vaccines AgainstShipping-fever and Pneumonic Pasteurellosis of Cattle, Sheep, and GoatsBased on In-frame Clean Deletions of the Leukotoxin Structural Gene ofPasteurella haemolytica

Materials and Methods

Mutagenesis of Plasmid Origin of Replication.

A 1.2 kb DNA fragment containing the putative pD80 origin of replicationwas amplified by PCR using the 4.3 kb ampicillin-resistance plasmidisolated from P. haemolytica serotype 1 strain NADC-D80 as template (1).Forward primer 5′-CCG GAT CCC CAA TTC GTA GAG GTT TC-3′ (SEQ ID NO:1)and reverse primer 5′-CCG GAT CCG CTG AAA GCG GTC GGG GG-3′ (SEQ IDNO:2) were used. The product was cloned into pCR2.1 vector (Invitrogen,San Diego Calif.) using the manufacturer's directions. Akanamycin-cassette was prepared by passage of pBC SK (Stratagene, LaJolla Calif.) containing a derivative of the Tn903 kanamycin gene(Pharmacia Biotech, Piscataway N.J.) cloned into the unique EcoRI sitethrough the E. coli strain PhaImtase to protect against PhaI-cleavage.

The cloned 1.2 kb insert (1 μg) was excised from pCR2.1 by EcoR1digestion and ligated overnight to the EcoR1-digested PhaI-methylatedkanamycin cassette (0.25 μg). The ligation mixture was concentrated byEtOH precipitation and electroporated (Gene-pulser, Bio-Rad, RedfieldCalif.) into P. haemolytica serotype 1 strain NADC-D153 using 18 kv/cmand 1000 W.

Plasmid DNA was obtained from a kanamycin-resistant transformant whichwas 2.5 kb in size and cleaved into fragments of 1.2 and 1.3 kb whensubjected to EcoR1. One μg of the plasmid was mutagenized withhydroxylamine for 1 hour at 65° C. as previously described (2).

Selection of Temperature-sensitive Plasmid Origin of Replication.

The mutagenized plasmid was dialyzed overnight at 4° C. against TE,concentrated by ethanol precipitation, and electroporated into freshNADC-D153 as described above. After 2 h recovery in Columbia broth(Difco Laboratories, Detroit Mich.), the cells were plated onto 10Columbia blood agar base plates containing 50 μg/ml kanamycin and wereincubated at 30° C. After 20 h, the plates were moved to 40° C. for anadditional 6 h.

Colonies were selected which were atypically small and cloned to freshkanamycin plates and were incubated overnight at 30° C. Growth from theclones was duplicated onto plates with and without kanamycin andincubated overnight at 40° C.

Clones which failed to grow on selective media at 40° C. but grewwithout selection were presumed to be temperature-sensitive for eitherplasmid maintenance or for expression of kanamycin. Growth from plateswithout antibiotic selection at 40° C. was passed to selective plateswhich were incubated overnight at 30° C. Clones which exhibited light orno growth on this passage were presumed to contain temperature-sensitiveorigins of replication. These clones were passed from the original 30°C. selective plate to fresh selective plates and to selective broth. Theclones were rechecked by passage with and without selection at 40° C.and 30° C. Although each clone exhibited the correct phenotype, plasmidmini-preps from the broth cultures yielded small amounts of a 2.5 kbplasmid from only one of the cultures. The remaining clones were notexamined further.

Construction of Dual-origin Temperature-sensitive Shuttle Vector.

The temperature-sensitive origin of replication was excised from theabove plasmid by EcoRI digestion and then made blunt by treatment withKlenow fragment of DNA polymerase I and all four dNTPs. The fragment wasligated overnight at 4° C. with SmaI-digested pBC SK. The ligation mixwas used to transform E. coli DH 10-B (Life Technologies, GaithersburgMd.).

Plasmid containing a 1.2 kb insert was recovered from achloramphenicol-resistant colony. The plasmid was digested with SalI andligated to SalI-digested kanamycin cassette overnight at 4° C. Theligation mixture was electroporated into E. coli DH10-B and plated ontokanamycin 50 μg/ml. Plasmid recovered from a kanamycin-resistant colonywas digested with BssHII, made blunt as above, treated with calfalkaline phosphatase to remove the terminal phosphates, and ligatedovernight at 4° C. with an approximately 900 bp blunt fragmentcontaining the Co1E1 plasmid origin of replication.

The ligation mixture was electroporated into E. coli PhaImtase (1) andplated onto kanamycin-containing plates. Plasmid was recovered from akanamycin-resistant colony which yielded a single approximately 3.5 kbfragment with EcoR1-digestion and fragments of 2.2 and 1.3 kb withSalI-digestion. The plasmid was given the designation pBB80C. Theplasmid was electroporated into P. haemolytica to confirm thetemperature-sensitive origin of replication; it still supportedbacterial growth on kanamycin at 30° C. but not at 40° C.

Cloning and manipulation of lktA. A 3.15 kb EcoRV fragment of P.haemolytica genomic DNA containing lktC and approximately 75% of thelktA coding region was ligated into the EcoRV site of pBB80C. Theresulting plasmid was amplified in E. coli DH10-B and given thedesignation pBB80ClktA.

Plasmid pBC SK (0.25 μg, used to provide additional NaeI-sites in trans)was mixed with 0.25 μg pBB80ClktA and digested with NaeI for 18 h at 37°C. The resulting partially digested plasmid DNA was extracted withphenol-chloroform-isoamyl alcohol (PCI), precipitated with ethanol,ligated at 4° C. overnight, then re-extracted and precipitated. Theligation mixture was digested with PvuII, which cleaves both pBC SK andthe 1035 bp NaeI fragment internal to LktA.

Five ng of the digested DNA was electroporated into E. coli PhaImtaseand plated on Columbia blood agar base plates containing 50 μg/mlkanamycin. Plasmid DNA from selected transformants was screened bydigestion of plasmid minipreps with EcoRV alone or together with NgoM1(an isoschizomer of NaeI). A clone containing a 1035 bp deletion wasselected and given the designation pBB80CΔlktA.

Recovery of Leukotoxin Mutants.

Plasmid pBB80CΔlktA was electroporated into fresh P. haemolytica strainNADC-D 153 at 18 kv/cm and 1000 W. The cells were allowed to recover 2hours at 30° C. in 1 ml Columbia broth, then were plated 100 μl/plate onColumbia agar plates containing 50 μg/ml kanamycin. After 48 hincubation at 30° C., four colonies were passed to kanamycin platescontaining 5% defibrinated bovine blood and were incubated overnight at37° C. to select for single-crossover mutants. Four colonies from the37° C. passage, 2 hemolytic and 2 non-hemolytic from each originaltransformant (16 total), were passed to Columbia broth without selectionand incubated overnight at 30° C. to resolve the single-crossovermutations.

Growth from the 30° C. Columbia broth was struck for isolation ontoblood agar base plates containing 5% defibrinated bovine blood andincubated overnight at 37° C. Growth was also passed to fresh Columbiabroth successively for a total of 4 passages at 30° C. to furtherascertain the rate at which kanamycin resistance was lost at 30° C.without selection. Isolated colonies from the first 30° C. passage wereduplicated in an array on selective and on non-selective platescontaining 5% defibrinated bovine blood.

A kanamycin-sensitive clone which demonstrated no detectable hemolyticactivity on the non-selective plate was selected for further study.Additional strains of P. haemolytica obtained from the repository at theNational Animal Disease Center were later subjected to similar treatmentas above. These strains were isolated from pneumonic lung and included:NADC D632, ovine serotype 1; NADC D121, ovine serotype 2; NADC D 110,ovine serotype 5; NADC D174, bovine serotype 6; NADC D102, ovineserotype 7; NADC D844, ovine serotype 8; NADC D122, ovine serotype 9;and NADC D712, ovine serotype 12.

Characterization of the Putative Leukotoxin Mutant.

To define the chromosomal deletion, DNA was amplified from whole cellsof the putative leukotoxin mutant and from its parent strain NADC-D 153by PCR, using primers nested within the EcoRV termini of the original3.15 kb EcoRV genomic fragment. The products were electrophoresed on a1.2% agarose gel both intact and after NgoM1 digestion.

To determine leukotoxic activity, log-phase culture supernatants fromthe putative mutant and its parent were prepared from Columbia broth 3hour cultures. Two-fold dilutions of the supernatants were assayed usingBL-3 target cells and MTT dye (7).

To determine the expression of the putative altered leukotoxin product,the culture supernatants, as well as a third culture supernatant fromour original leukotoxin deletion mutant which produces no detectableleukotoxin, were concentrated approximately 15-fold using 30,000 mwultrafilters (Centriprep, Amicon, Beverly, Mass.). The retentate waselectrophoresed in duplicate on SDS-PAGE, and one gel was stained usingCoomasie blue.

The second gel was blotted onto a nylon membrane for Western blotanalysis. The membrane was washed, probed with anti-leukotoxinmonoclonal antibody 601 (provided by Dr. S. Srikumaran, Lincoln Nebr.),labeled with anti-mouse IgG alkaline phosphatase-conjugated antibody(Sigma), and stained with nitro blue tetrazolium (Sigma).

Pasteurella haemolytica mutants of other than NADC D153ΔlktA serotype 1were characterized by PCR analysis and growth characteristics on bloodagar plates only. Their production of altered leukotoxin protein was notconfirmed.

Results

The temperature-sensitive origin of replication derived from theendogenous P. haemolytica ampicillin-resistance plasmid proved to be auseful tool for the construction of deletion mutants in that organism.The origin of replication was assumed to reside within a non-codingregion from nucleotides 3104 to 4293 of the native plasmid. The 1.2 kbPCR product of that region ligated to a 1.3 kb Tn903 kanamycin cassetteresulted in a 2.5 kb product capable of the stable transformation of P.haemolytica as evidenced by less than 1% loss of plasmid after 100generations in broth culture at 37° C. These data indicate thatessential replication functions reside within that 1.2 kb region of thenative plasmid.

Efficiency of transformation of P. haemolytica dropped about 10-foldafter hydroxylamine mutagenesis, indicating perhaps the DNA was notparticularly damaged. Nevertheless, 10 colonies which were atypicallysmall were recovered after 20 hours at 30° C. and 6 hours at 40° C. Twoof these colonies grew on selection at 40° C. and were discarded. Of theremaining 8 colonies, four were found to retain kanamycin-resistanceafter passage without selection at 40° C. These 4 colonies were presumedto contain plasmid which was temperature sensitive for the expression ofkanamycin-resistance and were also discarded.

The remaining 4 colonies, presumed to contain plasmid which wastemperature sensitive for maintenance, were recovered from the original30° C. plate and passed again to 40° C. and 30° C. Although each grewwell without selection at both temperatures, failed to grow withselection at 40° C., and failed to retain kanamycin-resistance after 40°C. passage, only one clone yielded sufficient plasmid for further studyby a rapid alkaline lysis procedure. It was assumed the other 3 coloniesalso contained plasmid, but the rapid plasmid purification procedurefailed to recover sufficient quantities to visualize on agarose gels.The plasmid yield from the positive clone was very low.

To facilitate subsequent isolation and cloning, a multiple-cloning siteand a Co1E1 origin of replication was added to the temperature-sensitivepD80 origin. The temperature-sensitive origin and a fresh kanamycincassette were placed within the multiple-cloning site of pBC-SK, thenthe vector backbone was replaced with a <1 kb copy of the Co1E1 origin.This approximately 3.5 kb plasmid, pBB80C, retains most of the uniquerestriction sites of pBC-SK, replicates efficiently in E. coli, andtransforms P. haemolytica at 30° C. with moderate efficiency. In P.haemolytica the Co1E1 origin fails to support replication, and plasmidmaintenance is dependent on the mutated pD80 origin. In this situation,pBB80C failed to support growth on selective media at both 37 and 40° C.but supported moderate growth at 30° C. (FIG. 1).

To introduce an in-frame deletion within the coding region of P.haemolytica lktA by allelic exchange, an EcoRV fragment containing partof the leukotoxin operon was cloned into pBB80C, yielding pBB80ClktA.The clone extended approximately 500 bp upstream from the lktC startcodon and included about 75% of lktA (FIG. 2).

Within the EcoRV fragment are two NaeI sites which cleave between codonswithin lktA leaving blunt termini (FIG. 3). The NaeI sites are situatednearly evenly 1 kb apart within the EcoRV fragment. Digestion ofpBB80ClktA with NaeI was complicated by the fact that NaeI is among agroup of restriction endonucleases which show a dramatic site preferencefor cleavage (4). This enzyme requires simultaneous interaction with twocopies of its recognition sequence before cleaving DNA. With certainenzymes of this type, the second copy may be supplied in trans, so itwas chosen in this experiment to supply additional recognition sites tothe digestion misture by adding pBC SK, which contains one site.Although this strategy resulted in incomplete cleavage after overnightdigestion, a 1 kb fragment was evident in the mixture, indicating bothNaeI sites had cleaved on some of the pBB80ClktA molecules.

Cleavage after ligation with PvuII, which is contained both in pBC SKand within the 1 kb NaeI fragment to be deleted, apparently eliminatedmost undesired products, because all transformants screened forpBB80CΔlktA contained the desired 1035 bp deletion. Each of theserecleaved with NgoM1, indicating the new NaeI site was intact and theproduct should be in-frame to the lktA start codon.

Pasteurella haemolytica transformed with pBB80CΔlktA required nearly 48hours to achieve good colony size at 30° C. Passage to 37° C. by simplystreaking heavily on a kanamycin-containing plate resulted in numerousisolated colonies, some hemolytic and some not. These results areconsistent with specific integration of the plasmid into the leukotoxinoperon (FIG. 4). Since the replacement plasmid contained intact operonsequence upstream from the deletion, including the promotor, upstreamsingle-crossover products were expected to express the entire operonnormally. Downstream single-crossover products, however, were expectedto contain two defective copies of lktA, since the C-terminal encoding25% of lktA was not present on the replacement plasmid. One copy of theleukotoxin gene therefore would be expected to contain the 1 kb deletionand the other copy a truncated C-terminus. Hemolytic activity haspreviously been shown to be correlated with expression of active LktA(3, 5, 6).

Passage of single-crossover products at 30° C. resulted in anunexpectedly low rate of plasmid resolution from chromosome. Previouswork with pBB192C, a temperature-conditional plasmid derived from the P.haemolytica streptomycin-resistance plasmid, exhibited 90 to 99%reversion to kanamycin sensitivity after a single passage at 37 or 30°C. respectively. In this experiment, of 80 isolated colonies testedafter one passage at 30° C., only two became sensitive to kanamycin. Oneof the two was non-hemolytic and was later shown to be adouble-crossover mutant (FIG. 5). Further passage increased thepercentage of kanamycin-sensitive CFU in non-selective cultures tonearly 50% after 4 passages. Many of these colonies exhibited anon-hemolytic phenotype and were probably double-crossover products.

To generate mutants of the other serotypes, 4-8 hemolyticsingle-crossover products were selected and passed at 30° C. for one ormore passages in broth. Growth was struck for isolation on each passage,and non-hemolytic colonies were selected for testing by PCR and growthon kanamycin-containing media. In each case, non-hemolytic colonieswhich were kanamycin-sensitive were confirmed by PCR to be deletionmutants containing single NaeI sites.

We assume that pBB192C contains a more robust origin of replication thandoes pBB80C, as evidenced by the relative amounts of plasmid recoveredfrom the respective cultures. If activity of an integrated plasmidorigin destabilizes chromosomal replication, it would be expected thatgreater instability would be realized as plasmid origin activityincreases. This could account both for greater resolving rates ofpBB192C at 30° C. than at 37° C. and for the lower rates of resolving ofpBB80C compared to pBB192. During construction of our first leukotoxindeletion mutant, a large number of single crossover products wereobtained using suicide replacement plasmid (3), which containedampicillin selection. Although both the homologous arms were similar inlength to those of the current experiment, passage for even 100generations resulted in no reversion to a hemolytic phenotype or loss ofampicillin-resistance. These data further indicate that it is theactivity of plasmid origin which destabilizes the single-crossoverproducts.

PCR products from the putative leukotoxin mutants and their parentstrains were found to be 2 kb and 3 kb in size respectively, indicatinga deletion had been introduced into their respective lktA. Digestion ofthe PCR products with NgoM1 revealed 2 bands of approximately 1 kb fromthe mutants and 3 bands of approximately 1 kb from the parent strains,indicating the deletions should be in-frame to LktA. Leukotoxin activityin culture supernatants against BL-3 target cells from the serotype Imutant was <1:2 compared to 1:1024 from the parent strain, indicating nodetectable activity.

A new protein of approximately 65 kDa was detected in the culturesupernatant of this mutant by SDS-PAGE, consistent with the predictedmolecular weight of the deleted product. By Coomasie staining, the newproduct exceeded the concentration of the native LktA protein producedby the parent strain grown and harvested alongside the mutant. Thesmaller size of this product may allow more rapid or economicalexpression of the gene. The product reacted with the neutralizingmonoclonal antibody 601 at an apparent molecular weight of 66 kDa (FIG.6). No reaction was observed at 101-104 kDa, the apparent molecularweight of the native product observed in the culture supernatant of theparent strain.

EXAMPLE 2 Assessment of Vaccine Efficacy in Small Ruminants AfterIntramuscular Injection of a Polyvalent Combination of P. haemolyticaSerotypes 5 and 6

Materials and Methods

Vaccination of Animals.

Four lambs (Columbia, approximately 25 kg) and six goats (Toggenburg,approximately 15 kg) were colostrum deprived and raised at the NationalAnimal Disease Center, Ames, Iowa. Two lambs and three goats wererandomly selected and vaccinated with 4×10⁷ CFU each of P. haemolyticaNADC D110ΔlktA and NADC D174ΔlktA (serotypes 5 and 6 respectively) in 1ml Earles Balanced Salt Solution (EBSS), pH 7.4. The suspension wasdelivered intramuscularly in the mid-cervical region. After three weeks,the animals were similarly revaccinated.

Ten days after the second vaccination all ten animals were challengedwith 8.5×10⁷ CFU each of the parent strains NADC D110 and NADC D174mixed in a total volume of 5 ml EBSS instilled intratracheally at thetracheal bifurcation with a catheter. The inoculum was chased with 5 mlsterile EBSS. Five days after challenge all surviving animals wereeuthanized and necropsied.

Bacteria.

Pasteurella haemolytica strains NADC D110 (serotype 5, ovine lungisolate) and NADC D174 (serotype 6, bovine lung isolate) were grownseparately in Columbia broth (Difco Laboratories, Detroit Mich.)approximately 3 hours to late log phase, about 2×10⁹ CFU/ml. Growth wasdiluted in EBSS 1:50 for the vaccine dose or 1:100 for the challengedose. The two strains were mixed in equal volume and kept on ice priorto animal inoculation.

Samples and Data Collection.

Sera were collected the day of the first vaccination, 2 weeks later, theday of challenge exposure, and the day of necropsy. Rectal temperatureswere recorded for 3 days after each vaccination and twice daily fromchallenge exposure to necropsy. Clinical scores were subjectivelyassessed on the same schedule as rectal temperatures, based on degree ofdepression and appetite. At necropsy, lung specimens from 1 to 3 gramsin weight were obtained from areas containing abnormalities, whenpossible, for bacterial enumeration. Swab specimens were obtained fromtrachea, kidney, and liver for bacterial isolation. Lung lesion volumeswere estimated for each lobe of the lung, including both consolidatedareas and those which appeared merely atelectic. Total lung lesionscores were expressed as a percentage where each lobe was adjusted foran approximation of its contribution to air exchange as follows: rightcranial lobe, 6%; right cranial half of the middle lobe, 5%; rightcaudal half of the middle lobe, 7%; right caudal lobe, 35%; accessorylobe, 4%; left cranial lobe, 4%; left middle lobe, 6%; and left caudallobe, 32%.

Sample Processing.

Sera were tested for P. haemolytica antibody by indirecthemagglutination (IHA) against serotypes 5 and 6 (all animals) and byleukotoxin neutralization (vaccinates only) using BL-3 cells and MTT dye(7, 8). Lung specimens were weighed, and EBSS was added to bring thetissue plus fluid volume to 10 times the weight. The specimens wereground to yield a homogenous suspension, and ten-fold dilutions weremade in EBSS.

The dilutions (100 μl) were spread onto blood agar base platescontaining 5% defibrinated bovine blood and incubated overnight at 37°C. Colonies exhibiting typical P. haemolytica morphology wereenumerated, and 20 representative colonies (where available) wereserotyped using specific antisera (9). Swabs were rolled onto one-thirdof fresh blood agar plates and then each side of a sterile loop was usedto semi-quantitatively streak for isolation onto the remaining thirdsconsecutively.

Results

No local reaction was palpable or visible following either vaccinationin any vaccinate. The first dose of vaccine elicited a febrile response,particularly in the sheep, which had a fever on day 2 and 3 which peakedat 40.3° C. on day 3. The second injection elicited no clinicalresponse.

Prior to vaccination, the animals had a low IHA titer against bothserotypes 5 and 6 of P. haemolytica (Table 1). After the firstvaccination, the vaccinates' titer increased over 8-fold against bothserotypes. No response was evident after the second dosage. Only aslight increase in antibody titer, about 50%, occurred after challengeexposure. The control animals' titer increased slightly, about double,prior to challenge exposure. Between the time of challenge exposure andnecropsy, the one surviving control sheep increased its titer againstboth serotypes by about 32-fold.

Leukotoxin neutralization titers in the vaccinates increased variably.Both lambs and two goats seroconverted (increased at least 4-fold) afterthe first vaccination; one of the animals also seroconverted to thesecond dose. One goat remained seronegative throughout the study.

Following challenge, none of the vaccinates had a fever at any time.They remained alert and eating all their food until necropsy. Thecontrol animals had a fever the day after exposure averaging 40.7° C.All control goats and 1 control sheep died overnight between the firstand second day after exposure. The remaining control sheep remainedfebrile, anorexic, and depressed until necropsy.

Inspection of the vaccine injection site at necropsy revealed nodetectable reaction in the muscle. Slight subcutaneous discolorationabout 1 cm in diameter due to hemorrhage was detected in both sheep andtwo of the three goats.

Lung lesion volume of the vaccinates, corrected for ventilation capacityof each lobe, averaged 3.5% (Table 2). One goat had 95% of its accessorylobe with moderately firm consolidation from which 1.3×10⁶ CFU/g(equally of serotypes 5 and 6) were recovered. The remaining lunglesions were soft, consistent with atelectasis.

Of 19 lung specimens quantitatively cultured, 5 yielded P. haemolytica.Two animals yielded no P. haemolytica from their lung. Two yielded from2×10³ to 7×10³ CFU/g from their right cranial lobes or cranial half ofthe middle lobe only. The animal with accessory lobe involvement alsoyielded 1×10³ CFU/g from the right caudal half of its middle lobe andmoderate growth from its tracheal swab. All other tracheal swabs fromvaccinates were culture negative, as were swabs from liver and kidney.

One sheep had tight adhesions of visceral to parietal pleura and to thepericardium ventrally on both right and left sides involving all lobes.This sheep contained only minor lesions of atelectasis and yielded only2×10³ CFU/g from its right cranial lobe; both other lobes cultured werenegative.

Lung lesion volume of the controls (corrected for ventilation capacityof each lobe) averaged 52% (Table 2). The four animals which diedcontained large amounts of fibrinous pleural effusion and fibrinouspleural adhesions. The lung lesions were firm or moderately firm, andemphysematous and/or crepitous areas were evident. The sheep whichsurvived until the time of necropsy contained about 100 cc pleuraleffusion and a large (about 250 cc) fibrous mass occupying the plerualspace over the right cranial and middle lobes.

The lung lesions consisted primarily of firm fibrinous consolidation inthis animal. Of 17 cultured lung specimens, all yielded P. haemolyticafrom as few as 2.5×10⁴ CFU/g to 4×10⁹ CFU/g. The geometric mean countfor the four animals which died acutely was 2.5×10⁸ CFU/g; the survivingsheep had a mean count of 2.5×10⁵ CFU/g. Tracheal swabs from the fouranimals which died yielded heavy growth of P. haemolytica. The survivingsheep yielded light growth from its trachea. Liver swabs of all four andkidney swabs of two of the animals which died yielded P. haemolytica.The surviving sheep was culture negative in both liver and kidney.

Serotyping of isolates from lung revealed that the few coloniesrecovered from vaccinates were of serotype 5, except for the activelyinfected accessory lobe of one goat which yielded equal amounts of bothserotype 5 and 6. Control animals tended to yield a mixture of serotypesfrom each lobe, but the mixture varied widely from lobe to lobe in theanimals which died acutely (e.g. 95% of serotype 5 in the right craniallobe to only 5% of serotype 5 in the right caudal lobe). Isolatesrecovered from kidney or liver tended to be homogenous with respect toserotype in any given animal, but two animals contained serotype 5 inthese tissues, and the other two contained serotype 6.

The first dose of vaccine can induce a febrile response. The lack of afebrile response and immune response from the second dose implies thatsubstantial immunity is conferred by the first dose. The second dose wasapparently quickly dealt with by the immune system and did not developsufficient antigenic mass to elicit an anemnestic response. The dosageof organisms delivered in the vaccine (nearly 10⁸ CFU) may have exceededthat necessary to confer sufficient immunity. Modified-live vaccinestypically would be delivered at a lower dose, perhaps 10⁵ to 10⁷ CFU.The failure of the second dosage of vaccine to stimulate furtherantibody, as measured by IHA, may indicate that two doses wereunnecessary and that a single dose would have been sufficient.

The reactions observed at the vaccine injection sites were extremelyminor and did not involve muscular tissue, consistent with findingsusing leukotoxin negative mutants of serotype 1 in cattle. Thiscontrasts greatly with the response of leukotoxin positive strains givenintramuscularly to cattle, which evidence large swellings and necrosisin the area, often opening through the overlying skin. It is likely thatlittle or no local adverse reaction would occur with subcutaneous orintradermal vaccination, an alternative that may also tend to reduce thefebrile response to vaccination.

Thus polyvalent intramuscular vaccine elicited marked immunity in sheepand goats against polyvalent challenge. Adverse reactions were limitedto a febrile response after injection which might be controlled byreduced vaccine dosage or an alternative route of administration.

EXAMPLE 3 Assessment of Vaccine Efficacy in Cattle After OralAdministration and After Intramuscular Injection

Materials and Methods

Vaccination of animals.

Sixteen dairy-type calves, approximately 150 kg, were obtained from alocal dairy and housed at the National Animal Disease Center, Ames,Iowa. The calves were randomly assigned to one control group of six andtwo vaccinate groups of 5. Each group was separately housed undersimilar conditions to prevent spread of vaccine organism between groups.

To each calf in one group of vaccinates was subcutaneously administeredin the mid cervical region 1 ml of EBSS containing 1×10⁷ CFU P.haemolytica serotype 1, NADC D153ΔlktA in-frame deletion mutant on day0. These calves were revaccinated similarly with 7.0×10⁶ CFU in 1 mlEBSS on day 21. The other group of vaccinates was fed a pelleted ration(Growena, Ralston Purina, St. Louis Mo.) onto which 50 ml total volumeof a fresh broth culture containing 1×10⁹ CFU/ml NADC D153ΔlktA in-framedeletion mutant was poured on day 0. The calves were similarly fed 50 mlof 7×10⁸ CFU/ml on day 21.

On day 28 all calves were challenged intratracheally with 25 ml of theparent P. haemolytica in EBSS at 2×10⁷ CFU/ml using a catheter placed atthe tracheal bifurcation. The challenge was chased with 25 ml sterileEBSS. Calves which survived challenge were euthanized 4 or 5 days afterchallenge and necropsied.

Bacteria.

Pasteurella haemoltica strain NADC D153 and its leukotoxin mutant weregrown in Columbia broth approximately 2.5 hours to mid log phase, about1×10⁹ CFU/ml. Growth was diluted 100-fold in EBSS for injection or50-fold for challenge. Growth was used unwashed and undiluted for oraladministration. All preparations were kept on ice prior to animalinoculation.

Samples and Data Collection.

Sera were collected 3 days prior to the day of the first vaccination, 3weeks later, the day of challenge exposure, and the day of necropsy.Rectal temperatures were recorded for 3 days after each vaccination andtwice daily from challenge exposure to necropsy. Clinical scores weresubjectively assessed on the same schedule as rectal temperatures, basedon degree of depression and appetite.

At necropsy, lung speciments were obtained and treated as described inExample 2, above.

Sample Processing.

Sera were tested for P. haemolytica antibody by IHA against serotype 1and by leukotoxin neutralization using BL-3 cells and MTT dye. Lungspecimens were weighed, and EBSS was added to bring the tissue plusfluid volume to 10 times the weight. The specimens were ground to yielda homogenous suspension, and ten-fold dilutions were made in EBSS. Thedilutions (100 ml) were spread onto blood agar base plates containing 5%defibrinated bovine blood which were incubated overnight at 37° C.Colonies exhibiting typical P. haemolytica morphology were enumeratedand, where available, 10 representative colonies were serotyped usingspecific antisera. Swabs were rolled onto one-half of fresh blood agarplates, and then each side of a sterile loop was used tosemi-quantitatively streak for isolation onto the remaining two quartersconsecutively.

Results

No local reaction was palpable or visible following either parenteralvaccination. None of the calves exhibited a febrile response after thefirst parenteral or oral vaccination. One parenterally vaccinated calfexhibited a transient (1 day) fever of 40.4° C. after the second dose;no adverse reaction was noted with any of the remaining calves.

Prior to vaccination, the animals had a low IHA titer against serotype 1P. haemolytica (Table 3). After the first vaccination, the antibodytiter in calves fed the vaccine increased at least 8-fold over theirprevaccination titers. The second oral dose did not increase, and insome cases titers dropped 2-fold. Titers of parenterally vaccinatedcalves increased only about 2-fold after the first dose of vaccine,during which time similar titer increases occurred in the controlcalves. The second dose of parenteral vaccine elicited additionalantibody response in the parenteral vaccinates, seroconverting (4-foldincrease) 3 of these 5 calves.

Leukotoxin neutralization titers were relatively high in most calvesprior to vaccination (Table 3). Two orally vaccinated calvesseroconverted after the first vaccine dose. One parenterally vaccinatedcalf seroconverted after the second vaccine dose. Overall,antileukotoxin titers increased in both vaccinated groups on successivebleedings. Antileukotoxin titers of control calves tended to decrease onsuccessive bleedings.

Following challenge, some but not all of the parenteral vaccinatesexhibited fevers under 41° C.; the oral vaccinates remained afebrile.All the vaccinates remained alert and on-feed. One control animal diedthe third day after challenge. Another was euthanized on day 3 nearlymoribund. Two of the remaining control calves were depressed andoff-feed and maintained a fever until euthanasia on day 4 or 5. One ofthese calves was recumbent and thumping at the time of euthanasia. Theremaining 2 control calves became afebrile the third day afterchallenge. They resumed eating and were deemed alert.

Lung lesion volume, corrected for ventilation capacity of each lobe,averaged 4.4% for orally vaccinated animals, 7% for those subcutaneouslyvaccinated, and 32% for unvaccinated controls (Table 4). Lung lesions ofboth vaccinated groups were predominantly soft, consistent withatelectasis. Localized areas of firm consolidation were noted in 2 ofthe orally vaccinated calves and 4 of the parenteral vaccinates, withlimited pleuritis and moderate pleural adhesions in two animals of eachgroup. These firm areas were confined to fractions of single lung lobesin each case. Unvaccinated controls had multiple lung lobes whichcontained a substantially higher percentage involvement with firm,fibrinous consolidation associated with edema and extensive fibrinouspleuritis. Three of the six control animals contained a large amount ofpleural effusion.

Bacterial culture of lung specimens showed that 2 orally vaccinatedcalves and 1 parenterally vaccinated calf were culture negative in alltested lobes. The remaining vaccinates tended to have one or twospecimens which yielded substantial amounts of P. haemolytica, up to5×10⁷ CFU/g. The remaining lobes were either culture negative orcontained low amounts of P. haemolytica, about 10³ CFU/g. Unvaccinatedcontrol animals yielded multiple specimens with high numbers of P.haemolytica, over 10⁷ CFU/g with many between 10⁹ and 10¹⁰ CFU/g. Nasalswabs yielded P. haemolytica from 1 parenterally vaccinated calf and 4control calves. Tracheal swabs were culture-positive for P. haemolyticain 4 control calves, 1 of which was nasal culture-negative. Pleuralfluid was culture positive in 3 control calves. All vaccinates wereculture-negative from trachea and pleural fluid. No P. haemolytica wererecovered from liver or kidney of any calf. All P. haemolytica wereβ-hemolytic, and those tested were serotype 1.

Thus, vaccination with the modified-live P. haemolytica protectedagainst virulent challenge, whether the vaccine was administeredsubcutaneously or orally after top-dressing feed.

Adverse reactions to vaccination were limited to one animal exhibiting atransient fever after the second subcutaneous injection of vaccine. Nolocal irritation or swelling was evident nor any postmortemabnormalities at the injection site, and no clinical abnormalities werenoted in any animal, whether injected or fed vaccine. The vaccine dosageused for injection was about 4-fold lower than that used in Example 2,above.

Seroconversion by IHA was impressive for animals orally vaccinated. Allanimals' titer increased at least 8-fold after the first exposure. Lessimpressive was seroconversion after subcutaneous injection. No animalsseroconverted after the first dose, and only 3 of 5 sevoconverted afterthe second dose. The IHA procedure has been found useful as a measurefor animals' prior experience with P. haemolytica of specific serotypes(10-13). Its utility for predicting resistance to disease is unclear,however (14-16). While some researchers find a correlation between IHAtiters and disease, others find none. If one assumes that theserotype-specific antigens employed in the IHA procedure are not thoseinvolved in humoral protection, the discrepency can be explained.Vaccination could elicit an IHA response without significant protectionor, conversely, elicit little IHA response but substantial protection.In either case, it is not surprising that oral exposure would elicit agood response, assuming that such exposure is sufficient to qualify as“prior experience.” The subcutaneous vaccination, while apparentlyeffective, elicited a relatively minor IHA response. Our priorexperiment in small ruminants using IM injection resulted in substantialIHA responses to both P. haemolytica serotypes 5 and 6. Perhaps theroute of exposure directed the former to a primarily cell-mediatedresponse and the latter to a more humoral response.

Antileukotoxin titers were not impressive in either group, as only 3 of10 vaccinated animals seroconverted after vaccination. Antileukotoxintiters were substantial prior to vaccination, however, and may havecontributed to a decreased response. These preexisting titers may havebeen due to previous colonization by serotype 2 P. haemolytica, the mostcommon commensal P. haemolytica in calves' nasal passages.Alternatively, it is possible that replication of P. haemolytica aftervaccination was not great, perhaps because the bacteria were readilyhandled by the immune system, and therefore little antigenic mass ofleukotoxin was elaborated. Finally, there is the possibility that thealtered leukotoxin protein, although designed to leave immunodominantepitopes, is not particularly adept at stimulating a neutralizingresponse even if it is immunogenic. There is little doubt, however, thatsome leukotoxin neutralizing antibody was produced in response tovaccination.

The multiple large areas of firm lung consolidation in unvaccinatedanimals at necropsy and the relatively large concentration of P.haemolytica in those areas indicate an active infection which spreadfrom the initial site of inoculation. In contrast, the vaccinates(except the 3 with essentially clean, culture-negative lungs) hadrelatively smaller areas of consolidation confined to single lung lobeswhich contained moderately high numbers of P. haemolytica. Other lobesof these animals were either culture-negative or contained low tomoderate numbers of bacteria. These data may indicate that the infectionwas active primarily at the site of inoculation and bacteria were havingdifficulty establishing in other portions of the lung. The cultureresults from tracheal specimens might support that conclusion, since 4of the 6 control animals but none of the vaccinates yielded P.haemolytica from this source, which indicates the infection was not wellcontained in most of the controls.

The data are clear that both subcutaneous administration and oraladministration of the modified-live vaccine were of significant benefitto animals intratracheally challenged with wild-type P. haemolyticaserotype 1. Manipulation of dosage or use of intramuscular injectionsmight further improve the efficacy of parenterally administeredvaccines.

The orally administered vaccine was markedly efficacious. The necessarydose in this case is likely some threshold level which is sufficient tocause colonization of the upper respiratory tract or palatine tonsils.Although conceivable, it is unlikely the dosage was effective due topassage into the gastrointestinal system. Even 10¹⁰ CFU of P.haemolytica passing into the rumen would be a relatively small number oforganisms, and the possibility that these bacteria could compete againstthe rumen or intestinal flora and multiply is remote. Still, if the gutwere to respond and there is a mucosal immune system link in cattle, onemight expect the response to be beneficial. These possibilities might beinvestigated using genetically marked P. haemolytica such as arifampicin-resistant strain for which colonization can be detected withgreat sensitivity (7, 11).

The theory behind the oral vaccine is that animals naturally infectedwith P. haemolytica serotype 1 develop resistance to subsequent nasalcolonization by serotype 1 organisms. They also develop systemicantibodies against P. haemolyica and, variably, against leukotoxin. Anavirulent organism which is proficient at colonization of nasal passagesor palatine tonsils might elicit similar resistance or resistance topulmonary challenge without the possibility of causing pneumonicpasteurellosis.

It is even possible that passive protection might occur in some cases bycompetitive exclusion of virulent P. haemolytica. Delivery by carriageon feedstuffs is possible because the palatine tonsils sustain long-termcolonization by P. haemolytica (18, 19). These sites also are in thepath of incoming feed. Often course feedstuffs such as hay stems arefound within the larger sinuses of the palatine tonsils, indicating thatexposure to feed is significant.

We conducted preliminary experiments to test the ability of feed todeliver P. haemolytica to palatine tonsils or nasal passages using arifampicin-resistant strain of P. haemolytica. Calves fed infected feedbecame colonized in both tonsils and in nasal passages.

In summary, protection against virulent challenge was conferred bysubcutaneous or oral administration of a modified-live P. haemolyticavaccine. In this experiment, oral administration elicited greaterantibody responses and slightly greater protection. An additionalpotential benefit of vaccination via feed is that calves would not needto be caught to be vaccinated, thereby reducing stress for both the calfand the operator. A potential caveat is that at least some calves musteat or at least browse through the inoculated feed to become colonized.Calves which do not partake of the feed may later become immune afterexposure to calves which did partake.

EXAMPLE 4 Preliminary Assessment of Safety and Efficacy ofOrally-administered Vaccine for Calves Already in Typical MarketingChannels

This experiment was designed to test the efficacy of an experimentalpulmonary vaccine produced by personnel at Texas A & M University.Within that experiment, and balanced between the groups of calvesutilized by Texas A & M, was our smaller experiment involving 18 head ofcalves. Our experiment was designed to see if feeding our vaccine strainto calves in the early stages of typical marketing channels would resultin colonization, elicit an immune response, and possibly reduce theincidence of shipping fever.

A field experiment was conducted in the Fall of 1997 with 105 steercalves (average 207 kg) procured from local sales barns by anOrder-Buyer in eastern Tennessee. Although the primary objective of theexperiment was to test an experimental vaccine by Texas A&M University,18 calves were fed the in-frame leukotoxin mutant 4 days prior toshipment to a feedlot in Texas about 1600 km away. The day afterpurchase, the calves arrived at an order-buyer barn where they wereear-tagged, vaccinated against clostridia, infectious bovinerhinotracheitis, and parainfluenza-3 virus, wormed with ivermectin, andcastrated by banding. Blood was collected for serum, rectal temperatureswere recorded, and nasal mucus specimens were collected.

Odd numbered calves were vaccinated with the experimental Texas A&Mpreparation. Nine odd- and nine even-numbered calves were separated intoa pen approximately 200 by 400 which contained a 120 feed bunk and asource of fresh water. A suspension of P. haemolytica NADC-D153ΔlktA(100 ml) was poured onto 35 kg of a commercial calf ration (Growena,Ralson Purina, St. Louis Mo.) and 15 kg of fresh grass hay. The bacteriawere grown on 10 Columbia agar plates overnight at 37° C. afterspreading inoculum for confluent growth. Growth was harvested into EBSSto a density approximating 2×10⁹ CFU/ml, and the resulting suspensionwas placed on ice until the calves were penned, whereupon 150 ml wastop-dressed onto the above feed.

Four days after feeding the vaccine, the calves were loaded onto a truckand transported to Bushland, Tex., where an experimental feedyard isoperated jointly by the USDA Agricultural Research Service and by TexasA&M University. Upon arrival the next day the calves appeared exhausted,as is typical of shipping this distance. The calves were run through thechute and rectal temperatures were recorded. The calves were then sortedinto 6 groups and allowed to rest overnight. The next day, the calveswere again run through the chute. Blood and nasal mucus was collected,rectal temperatures were recorded, and weights were taken. Many of thecalves were febrile (over 40° C.) with nasal discharge and loose stool.

The protocol called for treating calves for shipping fever withantibiotic on the second consecutive day of fever using tilmicosin(Micotil, Eli Lilly, Indianapolis Ind.). Calves not responding within 2days of treatment were to be treated with long-acting tetracycline(LA-200, Pfizer Inc., New York N.Y.). It was deemed expedient,considering the number of hot calves, to run all calves through thechute daily for 4 days to record all rectal temperatures. Serum, nasalmucus, weights, and rectal temperatures were then collected weekly(counting from the day after arrival) for 4 weeks, as described above.

The second day after arrival, 55 calves were treated using tilmicosin.Additional calves were treated subsequently until 22 days after arrival,bringing the total number treated to 84% of surviving animals. Ten totalanimals died within 4 days of arrival, 6 given the Texas A&M product and4 non-vaccinates. No animals given the oral vaccine died.

Postmortem observations revealed fibrinous pneumonia in all ten deadanimals, and P. haemolytica was recovered from all lungs along with P.multocida in a few lungs. Serotyping of lung isolates revealed that 9calves died of pasteurellosis by serotype 1 and 1 calf by serotype 6. Nostatistically significant differences were noted in morbidity (as judgedby treatment) between the orally-vaccinated, Texas A&M-vaccinated, orcontrol animals (78%, 84%, and 87% respectively). Nor was the differencein mortality significant (11.5% of non-orally-vaccinated versus 0% oforally-vaccinated calves, p >0.05).

Antibody titers (measured by IHA against serotype 1 P. haemolytica)increased significantly (p<0.01) between samples taken at theorder-buyer barn and those taken on arrival at the feedyard for bothorally-vaccinated and Texas A&M vaccinated calves compared tonon-vaccinates. Overall, the calves gained 29 kg between purchase andthe termination of the experiment after 28 days in the feedyard. Onegroup, orally-vaccinated calves which did not receive the Texas A&Mvaccine, gained significantly more weight than any other group (p<0.01,n=9) at 40.2 kg. All other groups did not significantly differ in thisparameter.

Pasteurella haemolytica serotype 1 and, to a lesser extent, serotype 6were recovered from nasal mucus of most calves one or more times at thefeedyard. The groups did not differ significantly in shedding theorganism. Some but not all calves which received the oral-vaccine shedthe mutant organism in one or more nasal mucus specimens during thefirst week at the feedyard, indicating that the inoculum was sufficientto colonize their upper-respiratory tracts under these conditions.

This experiment demonstrates that our experimental oral vaccine can bedelivered in feed at an order-buyer barn prior to shipment to thefeedyard and thereby colonize and elicit an immune response within 1week. Morbidity and mortality in the current experiment were unusuallyhigh. In addition to frequent isolations of P. haemolytica, respiratorycoronavirus and P. multocida isolations were common. The number ofcalves from which coronavirus was isolated was unusually high and mayaccount for the unusually heavy morbidity and the frequent diarrheaobserved. The number of calves requiring retreatment was also unusual,suggesting that bacteria other than P. haemolytica played a significantrole in the outbreak.

Tilmicosin is an antibiotic with a narrow spectrum of activity, targetedand advertised primarily to combat P. haemolytica. Given that bacteriaother than P. haemolytica and viruses such as respiratory coronaviruswere prevalent, it is not particularly surprising that the monovalentvaccines against P. haemolytica did not significantly reduce morbidity.However, none of the orally-vaccinated calves succumbed to pneumonicpasteurellosis compared to 11.5% of the others, suggesting that thevaccine played a role in reduction of mortality. The substantiallygreater weight gain of calves given only the oral vaccine furthersupports the conclusion that the vaccine reduced disease in thesecalves. Administration of the Texas A&M product together with the oralvaccine may have resulted in a reduction in the response to one or bothproducts or in responses deleterious to disease-resistance and therebyreduced the benefit conferred by the oral vaccine alone.

EXAMPLE 5 Ability of the P. haemolytica Serotype 1 Leukotoxin In-frameDeletion to Colonize Nasal Passages of Calves Stressed byConcurrentbovine Herpes Virus Type 1 Infection

Pasteurella haemolytica serotype 1 is recovered sporadically inrelatively low amounts from nasal mucus specimens of normal healthycalves. After stress or respiratory viral infection, P. haemolyticaserotype 1 can proliferate explosively in nasal passages to become thepredominant flora. Very high amounts of bacteria are shed in nasal mucusof such calves. It is believed that these high numbers of bacteria areinhaled or aspirated into susceptible lung to result in pneumonicpasteurellosis. Thus, this experiment was designed to obtain preliminarydata on whether leukotoxin deletion mutants of P. haemolytica cancolonize nasal passages under these conditions and, if so, whether theymight competitively exclude colonization by wild-type P. haemolytica.Both serotype 1 and serotype 6 organisms were used because both areknown to cause fatal fibrinous pneumonia in calves.

Materials and Methods

Vaccination of Animals.

Eight crossbred dairy-type calves, about 150 kg, were purchased from alocal dairy and maintained at the NADC. The calves were separatedrandomly into 2 groups of 4 each such that no contact could occurbetween the groups. The calves were allowed to acclimate for 10 daysprior to initiation of the experiment.

Infectious bovine rhinotracheitis virus (Coopers strain, kindly providedby National Veterinary Services Laboratories) was aerosolized into eachcalf's nostrils on inspiration, according to instructions provided byNVSL for challenge, resulting in a final dosage of 10^(9.4) TCID₅₀/nostril. After exposure to virus, one group of 4 calves were fed apalatable feed concentrate onto which 10 ml/calf of a mixed suspensionof P. haemolytica D153ΔlktA and D174ΔlktA (serotypes 1 and 6respectively) at 2×10⁹ total CFU/ml was poured. The other group was feduninoculated ration.

Five days after exposure to virus, the fed group was exposed byintranasal injection to 1.5 ml/nostril of P. haemolytica (mixture asabove) at 2.7×10⁸ total CFU/ml. Six days after exposure to virus, allgroups were exposed by intranasal injection to a mixture of wild-type P.haemolytica D153 and D174 at 5×10⁸ total CFU/ml.

Sample Collection and Analysis.

Nasal mucus specimens were collected on the day of exposure to virus,and 3, 4, 5, 6, 7, and 10 days after virus exposure. Serum was collectedthe day of exposure and 10 days later.

On the tenth day after exposure to virus, all calves were euthanized,and the lungs were examined grossly. Rectal temperatures were recordeddaily from the day of exposure to virus until euthanasia. Serum wastested for antibody against both serotype 1 and serotype 6 P.haemolytica by IHA.

Nasal mucus was diluted in 10-fold increments and spread onto blood agarbase plates containing 5% defibrinated bovine blood. After overnightincubation, P. haemolytica were identified and enumerated, and 20representative colonies were serotyped by a rapid plate agglutinationprocedure.

Results

Most calves were febrile within 3 days of virus-exposure, and peakfevers occurred on day 4 at 40.5° C. Only 3 calves remained febrile morethan one week, and all became afebrile by 10 days after virus exposure.

All calves were culture-negative for P. haemolytica in nasal mucus theday of virus-exposure. One calf fed P. haemolytica leukotoxin mutantsshed non-hemolytic serotype 1 organisms in its nasal mucus specimensstarting 3 days after virus-exposure and continued to shed leukotoxinmutants until euthanasia. The remaining 3 fed calves remainedculture-negative for P. haemolytica until day 6, one day afterintranasal exposure to the mixture of leukotoxin mutants. These calvesshed non-hemolytic P. haemolytica on days 6, 7, and, with one exception,day 10 (Table 5). The calves not deliberately exposed to P. haemolyticauntil day 6 remained culture negative for the organism until day 7,whereupon they shed mixtures, with one exception on day 10, of serotype1 and serotype 6 hemolytic P. haemolytica.

Three animals exposed to mutant P. haemolytica seroconverted (4-fold orgreater increase in titer) to both serotypes 1 and 6 between the time ofvirus-exposure and euthanasia. The fourth animal had a two-fold titerincrease against both serotypes. The remaining animals either increased2-fold or maintained a constant titer during that period.

The lungs at postmortem were mostly unremarkable. Calf 30, unexposed toleukotoxin mutants, had firm consolidation throughout its right caudalhalf of the middle lobe with 5% involvement of the cranial half. Calves17 and 18 had minor lesions of consolidation involving 5% or less of 2and 3 lung lobes respectively. No abnormalities were noted in theremaining calves.

Pasteurella haemolytica leukotoxin mutants were capable of colonizingthe nasal passages of calves which were concurrently infected with IBRvirus. Such colonization did not prevent or even reduce experimentalsuperinfection with wild-type P. haemolytica. Judging by numbers of P.haemolytica shed in nasal mucus, it appears the leukotoxin mutants wereless robust in nasal colonization. The wild-type bacteria colonized atlevels about 10-fold higher than did the mutants whether by themselvesor together. Nevertheless, the leukotoxin mutants were able to maintaina substantial level of colonization even in the presence of wild-type P.haemolytica, indicating that the bacteria were still quite robust. Infact, mixtures of wild-type parent strains and leukotoxin mutants passedin vitro in Columbia broth for 100 generations resulted in a populationslightly enriched for leukotoxin mutants, indicating that the leukotoxinmutants compete very well with wild-type under those conditions. Whetherit is possible to superimpose infection with leukotoxin mutants in theface of substantial colonization by wild-type P. haemolytica is notknown. Perhaps the leukotoxin mutants maintained their infection becausethey already had a foothold in the nasopharynx.

Our previous work with P. haemolytica infections using an IBR virusmodel indicates that bacterial infecion of the nasopharynx(specifically, the palatine tonsils) does not necessarily translate intoexplosive colonization of the nasal passages. Some calves which wereknown carriers of P. haemolytica serotype 1 in the palatine tonsilsfailed to become colonized in the nasal passages even though the nasalpassages were susceptible, as demonstrated by intranasal inoculation.Other similar calves of probable but unconfirmed carrier status didbecome colonized under similar conditions. This seeming paradox,infection in the pharynx which may or may not extend into adjacentsusceptible nasal passages, is not easy to explain. Perhaps the ciliaryflow from nasal passages carries material both forward, out of thenares, and backwards into the oropharynx.

Serum antibody titers against both serotype 1 and serotype 6 increasedsubstantially in three of the calves fed leukotoxin mutants. Since thecalves were killed on day 10, little time was available for an immuneresponse in the 7 calves which did not colonize until day 6 or 7. It istherefore likely that feeding the organism elicited or at leastfacilitated and immune response prior to the detected nasalcolonization.

Both serotypes 1 and 6 were recovered from nasal mucus in high amountsfrom every calf, most often as a mixed infection with both serotypes. Intwo cases, by day 10 serotype 1 had outgrown serotype 6 to become thepredominant flora. In one case, serotype 6 became the predominant flora.These results suggest that serotype 6 is nearly equal in its ability tocolonize under the chosen conditions. Given observations that serotype 6strain NADC D 174 elicits severe pneumonia in calves after intratrachealinoculation, one would expect that respiratory disease would occur incalves under conditions in the field. In fact, serotype 6 P. haemolyticahas been recovered previously from nasal passages of calves in fieldtrials and from lungs of calves which succumbed to pneumonicpasteurellosis. While serotype 1 remains the most common isolate in bothnasal passages of stressed calves and from pneumonic lung, serotype 6makes up a significant percentage of P. haemoytica isolations from nasalmucus or lung (about 10%) under these conditions.

Thus, in-frame leukotoxin deletion mutants of P. haemolytica are capableof colonizing the nasopharynx of calves made susceptible with concurrentIBR virus infection. Such infection was not sufficient to preventcolonization by wild-type P. haemolytica. Feeding the leukotoxin mutantsto calves concurrently with IBR virus exposure allowed one calf tobecome colonized to a high level in its nasal passages and appeared toresult in seroconversion to P. haemolytica in 3 of 4 calves. Both P.haemolytica serotypes 1 and 6 are capable of explosive colonizationduring respiratory virus infection, and each can do so in the presenceof the other.

TABLE 1 IHA antibody titers against Pasteurella haemolytica serotypes 5and 6 and leukotoxin neutralization titers before and after vaccination.First dose of vaccine on day 0, 2^(nd) dose on day 21. All animalsintratracheally challenged with wild-type serotypes 5 and 6 on day 28. n= 5 per group. Day 0 Day 14 Day 28 Day 33 Serotype 5 Vaccinate 2.4 6.06.2 6.8 Control 1.2 1.8 2.6 8*  Serotype 6 Vaccinate 1.4 6.2 6.2 6.8Control 0.8 1.4 1.4 6*  Leukotoxin** Vaccinate 0.4 3.0 3.4 — *Onesurviving sheep, 4 animals died 2 days after challenge and were nottested. **Control animals were not tested

TABLE 2 Lung lesion scores and postmortem lung bacterial culture results5 days after intratracheal challenge with Pasteurella haemolyticaserotypes 5 and 6. (n = 5 for each group, numbers expressed ± 95%confidence interval) Geometric mean P. Percent lung lesions**haemolytica in lung Vaccinates  3.5 ± 2.8* 1.2 × 10¹ ± 0.9 × 10^(1*)Controls 52.1 ± 21.7 6.3 × 10⁷ ± 2.5 × 10¹  *Significantly differentfrom control values, p < 0.001 **Percentage involvement of each lobeestimated and multiplied by the lobes' contribution to overall airexchange.

TABLE 3 IHA antibody titers against Pasteurella haemolytica serotype 1and leukotoxin neutralization titers before and after vaccination. Firstdose of vaccine on day 0, 2^(nd) dose on day 21. All animalsintratracheally challenged with wild-type serotype 1 on day 28. (n = 6for controls and 5 each for vaccinated groups) Day Day −3 Day 21 Day 2832 or 33 IHA titer IM vaccinate 2.6 3.4 4.8 5.8 Oral vaccinate 3.0 7.67.0 7.6 Control 2.2 3.3 3.5  5.8* Leukotoxin** IM vaccinate 6.8 6.8 7.47.8 Oral vaccinate 6.6 7.8 7.4 8.0 Control 6.8 6.3 6.2  6.8* *Foursurviving calves, 2 animals died 3 days after challenge and were nottested.

TABLE 4 Lung lesion scores and postmortem lung bacterial culture results4 or 5 days after intratracheal challenge with Pasteurella haemolyticaserotype 1. (n = 6 for controls and 5 each for vaccinated groups,numbers expressed ± 95% confidence interval) Geometric mean P. Percentlung lesions*** haemolytica in lung IM vaccinate  7.0 ± 7.3* 1.8 × 10² ±0.7 × 10^(2*) Oral vaccinate  4.4 ± 4.5** 1.4 × 10² ± 0.6 × 10^(2*)Controls 32.0 ± 13.4 1.6 × 10⁶ ± 1.0 × 10²  *Significantly differentfrom control values, p < 0.01 **Significantly different from controlvalues, p < 0.02 ***Percentage involvement of each lobe estimated andmultiplied by the lobes' contribution to overall air exchange.

TABLE 5 Shedding of P. haemolytica in nasal mucus of calves infectedwith IBR virus on day 0. Day 6 Day 6 Day 10 Calf* Phenotype** CFU/ml %St-1† CFU/ml % St-1 CFU/ml % St-1 15 mutant 4.0 × 10⁷ >95 4.0 × 10⁶ 501.0 × 10⁸ >95 wild-type none — 1.5 × 10⁸ >95 2.0 × 10⁸ 80 19 mutant 5.6× 10⁷   85 1.1 × 10⁷ 65 none — wild-type none — 1.1 × 10⁸ 10 5.0 × 10⁸<5 28 mutant 4.3 × 10⁷   80 2.5 × 10⁷ 70 1.2 × 10⁷ 60 wild-type none —1.2 × 10⁸ 55 1.9 × 10⁸ 20 29 mutant 1.6 × 10⁷ >95 2.0 × 10⁶ >95 4.0 ×10⁶ >95 wild-type none — 6.0 × 10⁷ 15 1.3 × 10⁸ >95  5 wild-type none —2.0 × 10⁸ 60 1.5 × 10⁸ 60 17 wild-type none — 1.5 × 10⁸ 50 4.1 × 10⁷ 4018 wild-type none — 2.0 × 10⁸ 10 2.0 × 10⁸ >95 30 wild-type none — 2.8 ×10⁸ 30 6.0 × 10⁸ 30 *Calves 15, 19, 28, and 29 exposed to serotype 5 and6 P. haemolytica leukotoxin mutants intranasally on day 5. All calvesexposed to wild-type P. haemolytica serotypes 5 and 6 on day 6.**Leukotoxin mutants are non-hemolytic; wild-type displays β-hemolysis.†20 representative colonies serotyped, when available.

REFERENCES

1. Briggs R. E., Tatum F. M., Casey T. A., Frank G. H. Characterizationof a restriction endonuclease, PhaI, from Pasteurella haemolyticaserotype A1 and protection of heterologous DNA by a cloned PhaImethyltransferase gene. Appl. Environ. Microbiol. 60:2006-2010. 1994.

2. Thomas C. M. Plasmid replication. In: PLASMIDS: A PRACTICAL APPROACH.K. G. Hardy, ed. IRL Press Limited, Oxford, England. 1987.

3. Tatum F. M., Briggs R. E., Sreevatsan S. S., Zehr E. S., Ling HsuanS., Whiteley L. O., Ames T. R., Maheswaran S. K. Construction of anisogenic leukotoxin deletion mutant of Pasteurella haemolytica serotype1: characterization and virulence. Microb. Pathog. 24: 37-46, 1998.

4. Conrad M., Topal M. D. Modified DNA fragments activate NaeI cleavageof refractory DNA sites. Nucleic Acids Res; 20:5127-5130. 1992.

5. Murphy G. L., Whitworth L. C., Clinkenbeard K. D., Clinkenbeard P. A.Hemolytic activity of the Pasteurella haemolytica leukotoxin. Infect.Immun. 63:3209-3212. 1995.

6. Fedorova N. D., Highlander S K. Generation of targeted nonpolar geneinsertions and operon fusions in Pasteurella haemolytica and creation ofa strain that produces and secretes inactive leukotoxin. Infect. Immun.65:2593-2598. 1997.

7. Briggs R. E., Frank G. H., Zehr E. S. Development and testing of aselectable challenge strain of Pasteurella haemolytica for studies ofupper-respiratory colonization of cattle. Am. J. Vet. Res. 59: 401-405,1998.

8. Frank G. H., Smith P. C. Prevalence of Pasteurella haemolytica intransported calves. Am. J. Vet. Res. 44:981-985. 1983.

9. Frank G. H., Wessman G. E. Rapid plate agglutination procedure forserotyping Pasteurella haemolytica. J. Clin. Microbiol. 7:142-145.1978.10. Frank G. H., Briggs R. E., Loan R. L., Purdy C. W., Zehr E. S.Serotype-specific inhibition of colonization of the tonsils andnasopharynx of calves by Pasteurella haemolytica serotype A1 aftervaccination with the organism. Am. J. Vet. Res. 55: 1107-1110. 1994.

11. Frank G. H., Briggs R. E., Zehr E. S. Colonization of the tonsilsand nasopharynx of calves by a rifampicin-resistant Pasteurellahaemolytica and its' inhibition by vaccination. Am. J. Vet. Res. 56:866-869. 1995.

12. Frank G. H., Briggs R E., Loan R. W., Purdy C. W., Zehr E. S.Respiratory tract disease and mucosal colonization by Pasteurellahaemolytica in transported cattle. Am. J. Vet. Res. 57: 1317-1320. 1996.

13. Purdy C. W., Cooley J. D., Straus D. C. Cross-protection studieswith three serotypes of Pasteurella haemolytica in the goat model. Curr.Microbiol. 36: 207-211. 1998.

14. McVey D. S., Loan R. W., Purdy C. W., Richards A. E. Antibodies toPasteurella haemolytica somatic antigens in two models of the bovinerespiratory disease complex. Am. J. Vet. Res. 50:443-447. 1989.

15. Jones G. E., Donachie D. W., Sutherland A. D., Knox D. P., GilmourJ. S. Protection of lambs against experimental pneumonic pasteurellosisby transfer of immune serum. Vet. Microbiol. 20:59-71. 1989.

16. Schimmel D., Erler W., Diller R. [The significance of antibodies toPasteurella haemolytica A1 in the colostrum of cows and blood serum ofcalves]. Berl Munch Tierarztl Wochenschr 105:87-89. 1992.

17. Frank G. H., Briggs R. E. Colonization of the tonsils of calves withPasteurella haemolytica. Am. J. Vet. Res 53:481-484. 1992.

18. Frank G. H., Briggs R. E., and Debey B. M. Bovine tonsils asreservoirs for Pasteurella haemolytica: Colonisation, immune response,and infection of the nasopharynx. In: Pasteurellosis in ProductionAnimals (Workshop Proceedings, Australian Centre for InternationalAgricultural Research.) pp 83-88. 1992.

2 1 26 DNA Pasteurella cf. haemolytica 1 ccggatcccc aattcgtaga ggtttc 262 26 DNA Pasteurella cf. haemolytica 2 ccggatccgc tgaaagcggt cggggg 26

We claim:
 1. A temperature sensitive plasmid which replicates at 30° C.but not at 40° C. in P. haemolytica and which has a mutation locatedwithin nucleotides 3104 to 4293 of the 4.3 kb ampicillin-resistanceplasmid isolated from P. haemolytica serotype strain NADC-D80.
 2. Thetemperature sensitive plasmid of claim 1 which is the plasmid which hasbeen deposited at the ATCC with Accession No. 98895.